In the plasma of patients suffering from disseminated intravascular coagulation (DIC) and in the plasma of patients being treated by the thrombolytic therapy using urokinase or tissue plasminogen activators (t-PA), a plasminogen is activated to produce a plasmin. The resulting plasmin immediately forms a complex with an .alpha..sub.2 -plasmin inhibitor (.alpha..sub.2 PI: Aoki et al., J. Biol. Chem., 251, 5956-5965, 1976) at a ratio of 1:1 in circulating blood. The complex is the above PIC. Therefore, by measuring the PIC, it is possible to detect the activation of the plasmin. Recently, the PIC in the plasma is considered important in the diagnosis of DIC, as a molecular marker for monitoring the thrombolytic therapy, and so on. Accordingly, it is necessary to accurately and simply measure the amount of PIC in the blood or plasma.
As conventionally known methods for measuring PIC, there may be mentioned the following five methods. The first method uses two-dimensional crossed immunoelectrophoresis. The second method is a latex agglutination using polyclonal antibodies which recognize the necantigen of PIC. The third method is an enzyme immunoassay wherein a polyclonal antibody against the plasminogen and a polyclonal antibody against the .alpha..sub.2 -plasmin inhibitor, and one of them is immobilized and the other is labelled by an enzyme. The fourth method is an enzyme immunoassay wherein a monoclonal antibody which specifically recognizes the sites inhibiting the fibrinolysis action of the plasmin and existing in the .alpha..sub.2 -plasmin inhibitor, and a polyclonal antibody against a plasmin are used; one of them is immobilized and the other is labelled by an enzyme; and the plasma specimen is diluted 1200-fold for measurement. The fifth method is the latex agglutination wherein two or three types of monoclonal antibodies which recognize the necantigen of PIC are used.
The first method, however, had the defects of a low sensitivity and lack of quantitativeness. The second method had the defects that the PIC cannot be specifically measured. The third method had the defects that, while the sensitivity is good, it is difficult to stably obtain anti-serum; and the immunoreaction is a two-step process,and so the procedure is complicated to require a long time for the measurement. In the fourth method, the defects of the third method were remedied by using a monoclonal antibody against the .alpha..sub.2 -plasmin inhibitor and carrying out the immunoreaction in a single step. However, the fourth method did not solve the defects inherent to the enzyme immunoreaction, that is, the complicated procedure and the long time required for the measurement. Further, the fourth method had the defects that it required the step of diluting the specimen 1200-fold so as to enable the single-step immunoreaction. The fifth method did not exhibit different specificity and measuring sensitivity in comparison with those of the second method wherein a polyclonal antibody is used, and did not solve the problem of the inability of specifically measuring PIC.
The present inventors engaged in research to develop a method for simply and accurately measuring PIC with good reproducibility, and as a result discovered three types of monoclonal antibodies; a first monoclonal antibody (PIC-1) exhibiting reactivity with both PIC and a plasminogen, a second monoclonal antibody (PIC-2) exhibiting reactivity with both PIC and an .alpha..sub.2 -plasmin inhibitor, and a third monoclonal antibody (PIC-3) exhibiting specific reactivity with PIC, but not exhibiting reactivity with a plasminogen and an .alpha..sub.2 -plasmin inhibitor which are the component proteins of the PIC, and further discovered that when a combination of two or more of these monoclonal antibodies are used, it is possible to specifically determine rapidly and accurately the PIC in the plasma without a step of dilution of the plasma specimen and without suffering from the interference of a free plasminogen and a free .alpha..sub.2 -plasmin inhibitor present in the specimen. Therefore, the object of the present invention is to provide the above-mentioned novel monoclonal antibodies, hybridomas producing the above monoclonal antibodies, and an immunological determination method using the monoclonal antibodies.